EP2938746B1 - Trousse de sonde pour détecter une séquence nucléotidique cible à un seul brin - Google Patents

Trousse de sonde pour détecter une séquence nucléotidique cible à un seul brin Download PDF

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EP2938746B1
EP2938746B1 EP13831873.8A EP13831873A EP2938746B1 EP 2938746 B1 EP2938746 B1 EP 2938746B1 EP 13831873 A EP13831873 A EP 13831873A EP 2938746 B1 EP2938746 B1 EP 2938746B1
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probe
nucleotide sequence
hybr
target nucleotide
kit
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EP2938746A1 (fr
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Filippo Causa
Edmondo Battista
Anna ALIBERTI
Angela Maria CUSANO
Paolo Netti
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Fondazione Istituto Italiano di Tecnologia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • the present invention relates to a probe kit for detecting a single strand target nucleotide sequence.
  • amplification of the sample is required for all these assays in order to allow a sufficient detection sensitivity.
  • a first example of this strategy consists in molecular beacons (MB), single strand oligonucleotides with a stem-loop structure formed by 4 parts: a) a loop consisting of a region of 15-30 nucleotides complementary to the target sequence; b) a stem, i.e. a short duplex segment formed by 5-8 base pairs; c) a reporter which is generally bound to the 5' end and emits fluorescent light; d) a quencher bound to the 3' end, which absorbs the light radiation of the reporter.
  • MB molecular beacons
  • the stem-loop structure of the molecular beacon undergoes a spontaneous fluorogenic conformational change.
  • the molecular beacon design provides a mechanism for both molecular recognition and transduction of the hybridization events in one single step and thus dramatically accelerates the molecular detection process.
  • molecular beacons have been used for real-time quantitative determination of nucleic acids, for the construction of self-reporting oligonucleotide arrays, and even for analysis in vivo ( Li Y. Zhou X. and Ye D. Biochem. Biophys. Res. Communications 2008, 373, 457-461 ).
  • a second example of the nucleic acid displacement strategy consists in the double-stranded assay, in which a nucleotide sequence containing a fluorophore labelled on the 5' end is designed to be complementary to the nucleotide sequence of interest.
  • a complementary sequence is designed with respect to the fluorophore probe but with a shorter length and its 3' end is labelled with a quencher.
  • the fluorophore and quencher probes are in close proximity diminishing the fluorescence signal. With the target, the quencher probe is replaced due to the thermodynamically driven binding event between the fluorophore probe and the target. Therefore, the fluorophore is separated from the quencher and is able to fluoresce.
  • advantages of dsDNA probes include the possibility of adjusting the quencher-to-fluorophore ratio for noise minimization and the flexibility of modifying the lengths of the quencher sequence and the sticky end for improving the specificity, selectivity and kinetics of the assay.
  • these assays have generally been shown to be effective only for lengths longer than 50 bases.
  • Kit 10 per for detecting a single strand target nucleotide sequence 3 comprises at least one first nucleic acid probe 1 and at least one second nucleic acid probe 2.
  • both probe 1 and probe 2 are made of DNA.
  • Fluorophore 11 bound to the 5' end of probe 1 is preferably selected from the group consisting of FAM, TET, JOE, HEX, Oregon Green ® , TAMRA, ROX, Cy3, Cy3.5, Cy5, Cy5.5, CAL RedTM, Red 640, Cy5, and Cy5.5.
  • Quencher 23 of probe 2 is preferably selected from the group consisting of DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, and BHQ-3 and is compatible with fluorophores 11 bound to the 5' end of probe 1.
  • probe 1 and probe 2 are designed so that the following relation is met: ⁇ G hybr . target 3 ⁇ probe 2 > ⁇ G hybr . probe 1 ⁇ probe 2 , where:
  • probe 1 and probe 2 are designed so that 10 Kcal / mol > ⁇ G hybr . target 3 ⁇ probe 2 ⁇
  • probe 1 and probe 2 are even more preferably designed so that 35 Kcal / mol > ⁇ G hybr .
  • probe 1 and probe 2 are even more preferably designed so that 10 Kcal/mol >
  • this software uses the value of ⁇ G as a measure of the affinity between two nucleotide sequences, where the affinity represents the measure of the thermodynamic stability of the duplex formed by the two single strand oligonucleotides.
  • ⁇ H (enthalpy) represents the total energy exchange between the system and the surrounding environment (kcal/mole)
  • ⁇ S entropy
  • T represents the absolute temperature of the system in Kelvin degrees (Celsius + 273.15).
  • Target nucleotide sequence 3 preferably has a length from 15 to 100 bases, even more preferably from 20 to 40 bases.
  • Kit 10 allows to detect target nucleotide sequences 3 in a range of concentrations from 1 ⁇ 10 -11 M to 1 ⁇ 10 -22 M, i.e. in a very broad range. In particular, kit 10 allows to detect target nucleotide sequences 3 at concentrations from 1 ⁇ 10 -17 M to 1.10 -19 M, i.e. a very low concentrations.
  • probe 1, probe 2 and target nucleotide sequence 3 are shown.
  • probe 1 is x bases long (for example, 12 bases) and has a Cy5 molecule bound at the 5' end.
  • Probe 2 is x+y bases long (for example, 39 bases) and comprises, from the 5' to the 3' end, a first segment 21 which is x bases long (for example, 12 bases long) and complementary to probe 1, a quencher 23 (BHQ), and a second segment 22 which is y bases long (for example, 27 bases long) and complementary to at least part of target nucleotide sequence 3.
  • Target nucleotide sequence 3 is x+y bases long (for example, 39 bases long).
  • probe 1 and probe 2 In the presence of probe 1 and probe 2, these form a duplex having formation free energy ⁇ G hybr.probe1-probe2. In this situation, quencher 23 BHQ quenches the signal emitted by fluorophore 11 Cy5 and there is no fluorescence emission.
  • Probe 1 and probe 2 are designed on the basis of target nucleotide sequence 3 and their thermodynamic affinity is modulated so that the affinity of probe 2 for target nucleotide sequence 3 is higher than the affinity of the initial duplex between probe 1 and probe 2.
  • and the length of probe 1 are selected so as to optimize the displacement of probe 1 and the formation of the duplex between probe 2 and target nucleotide sequence 3.
  • kit 10 also comprises at least one microparticle 4 covalently bound to the 3' end of probe 1, preferably by means of an amide bond. Even more preferably, the bond between microparticle 4 and probe 1 is obtained by means of an appropriate amino-linker on probe 1.
  • multilayer particle 4 according to the present invention comprises:
  • First fluorophore 51 and second fluorophore 71 are different, first layer 5 and third layer -7 are not in contact with one another.
  • First fluorophore 51 and second fluorophore 71 can be selected from the group consisting of rhodamine, fluorescein, Cy2, Oregon Green, Alexa (488, 532, 546, 555) and others as long as the emission wave length do not overlap.
  • multilayer microparticle 4 also comprises:
  • Third fluorophore 91 is different from second fluorophore 71 and from first fluorophore 51, and third layer 7 and fifth layer 9 are not in contact with one another.
  • the third fluorophore can be selected from the group consisting of rhodamine, fluorescein, Cy2, Oregon Green, Alexa (488, 532, 546, 555) and others as long as the emission wavelengths do not overlap with the wavelengths of first and second fluorophore 51, 71.
  • Multilayer microparticle 4 preferably has a size from 0.5 ⁇ m to 2 ⁇ m.
  • Each layer of multilayer microparticle 4 preferably comprises esters and amides of acrylic acid or of methacrylic acid or vinyls or allyls, which are optionally substituted.
  • esters and amides of acrylic acid or of methacrylic acid or vinyls or allyls, which are optionally substituted there is also intended compounds equivalent thereto.
  • This definition also includes difunctional polymers used as cross-linkers such as, for example, bisacrylammide, polyethylenoxide-acrylate/-methacrylate etc.
  • Fluorophores 51, 71, 91 included in layers 5, 7, 9 may be used in the form of acrylates or methacrylates or vinyls or allyls with other chemical groups which allow the chemical bond to the polymer network of layers 5, 7, 9.
  • Figure 2B shows the process for the preparation of a specific multilayer microparticle 4.
  • a first layer 5 of polyethylene glycol dimethacrylate (PEGDA, 500 MW) and rhodamine B acrylate monomers is produced by free-radical precipitation polymerization.
  • a second layer 6 of polyethylene glycol dimethacrylate is produced around first layer 5 by seeded polymerization.
  • a third layer 7 of acrylic acid and fluorescein is produced around second layer 6 by seeded polymerization.
  • Different groups of microparticles with different fluorescence codes can be obtained by using different concentration ratios between rhodamine and fluorescein.
  • first layer 5 Microgels of polyethylene glycol dimethacrylate have been prepared by free-radical precipitation polymerization, using a concentration of total monomers of 1% (w/v). Polymerization has been performed in a 100 ml three-neck flask with round bottom, in which a filtered aqueous solution of monomers and 1% (w/v) polyvinyl alcohol (PVA) as surfactant have been added. This solution was heated to ⁇ 65 °C while being purged with N 2 gas and stirred vigorously for ⁇ 1 h. Then the reaction was immediately initiated by injection of a potassium persulfate (KPS) aqueous solution (to make a final KPS concentration of 0.06 % w/v).
  • KPS potassium persulfate
  • Second layer 6 The rhodamine-labelled microgel was resuspended in deionised water to a concentration of 10 mg/ml. These microgels were then used as seed particles, upon which a PEGDMA cross-linked layer was added.
  • a solution of rhodamine-labelled core microgels (100 mg, 10 ml) in deionised water (25 ml) was heated to 65 °C under a gentle stream of N 2 .
  • PEGDMA 240 mg was dissolved in water (10 ml), purged with N 2 at room temperature and then slowly added to the heated core solution.
  • the reaction was allowed to proceed for 6 h.
  • the microgels were dialyzed for 5 days, purified several times by centrifugation (for 15 minutes at 6500 rpm) and resuspended in deionised water to remove unreacted monomers, oligomers and surfactants, then stored at 4 °C prior to use until further use.
  • the conjugate between probe 1 and multilayer microparticle 4 was precipitated down by centrifugation at 6000 rpm for 15 min at room temperature. The supernatant was removed carefully with a pipette and the precipitant was resuspended in 1 ml Of Tris HCl, pH 8 buffer by agitating with a pipette tip and brief vortexing. This washing step was repeated three more times.
  • Example 2 microparticles with different ratios between first fluorophore 51 and second fluorophore 71
  • Polyethylene glycol (PEG) microgels were produced (particle size of about 1 ⁇ m).
  • the outer layer of these microparticles 4 was functionalised with carboxylic groups.
  • Two concentrations of fluorescein 71 (0.1 ⁇ m and 0.2 ⁇ m) were used for third layer 7, and three different concentrations of rhodamine 51 (0.1 ⁇ m, 0.01 ⁇ m e 0.005 ⁇ m) were used for first layer 5.
  • Six microgels were distinguished by means of a spectrofluorometer, on the basis of combinations of different concentrations of rhodamine 51 and fluorescein 71 in the production solution of multilayer microparticles 4 ( figures 7A and 7B ).
  • the approach for multilayer microparticle 4 production allows to define an univocal code on the particles in virtue of the fact that fluorophores 51, 71 are confined in space.
  • confocal microscope images were obtained of three microgels with different codes obtained by means of different concentration ratios between rhodamine 51 and fluorescein 71 respectively in first layer 5 and in third layer 7 of multilayer microparticle 4.
  • Figure 4 shows how the length of probe 1 influences the formation of the duplex between probe 1 and probe 2 of probe kit 10 according to the invention.
  • the results for a probe 1 of 8 bases and a probe 1 of 12 bases are shown.
  • 0.1 ⁇ M solutions of probe 1 (8 and 12 bases long) and probe 2 were reacted for 12 hours and fluorescence emission was evaluated.
  • the bond between probe 1 (8 bases long) and probe 2 is poorly stable as can be seen from the measurements of fluorescence relating to the formation of the duplex. In this case a very low loss in the percentage of fluorescence is registered.
  • the duplex between probe 1 (12 bases) and probe 2 results more stable as confirmed by a greater percent loss of fluorescence emission of the complex probe 1 - probe 2 if compared to sole reference probe 1.
  • ⁇ G The values of ⁇ G have been computed by means of the Oligocalc software.
  • Table 1 Probe name Sequence Length ⁇ G (Kcal/mol) HIV probes (on the basis of Genbank sequence:AF033819.3 positions 6520-6559) HIV first probe (tail-Cy5) 5' Cy5 ACT GCT GTT AAA C6 NH 2 -3' 12
  • HCV probes on the basis of Genbank sequence: M67463.1 positions 160-195) HCV first probe (tail-Cy5) 5' Cy5 TTC CGG TGT ACT-C6 NH2-3' 12
  • Genb on the basis of human coronavirus bank:AF304460 positions 16710-167 sequence 747) 229E, whole SARS first probe (tail-Cy5) 5' Cy5 GGC TCC AGT ATA -C6 NH2- 3' 12
  • probe 1 40 pmoles of probe 1 were mixed with 40 pmoles of probe 2 in Tris HCl, pH 8. Quenched samples were used as reference in order to evaluate the displacement efficiency.
  • target nucleotide sequences miR155 can be detected up to concentrations of 5 nM with a range from 50 nM to 5 nM and a LOD of 1 nM.
  • probe kit 10 is capable of capturing and distinguishing target nucleotide sequences 2 even within longer sequences (as would occur in an actual context, since target nucleotide sequence 3 would be within a gene), displacement experiments were carried out using the 99 base long nucleotide sequences shown in table 2. These experiments were carried out in homogeneous phase.
  • Table 2 Probe name Sequences Length (nt) HIV 100 R 99 HIV 100 M 99 HIV 100 L 99
  • the HIV 100 R, HIV 100 M and HIV 100 L probes were designed so that target nucleotide sequence 3 is respectively at the 5' end, in the middle and at the 3' end of the 99 base long sequence.
  • Example 7 heterogeneous phase assay (microparticle conjugated probes)
  • FIG. 3 diagrammatically shows the heterogeneous phase assay.
  • first probe conjugated with the microgel in 250 ⁇ l of Tris HCl hybridization buffer pH 8 was mixed with 350 pmoles of second probe (250 ⁇ l) . The mixture was incubated at room temperature overnight. The microgels were then washed with hybridization buffer and resuspended in 1 ml of buffer at a final concentration of 1 ⁇ g/ ⁇ l. 50 ⁇ l (50 ⁇ g) of quenched microgel were mixed to 450 ⁇ l of a solution containing target probe sequences 3 at different concentrations ranging from 10 -11 to 10 -22 M and incubated at room temperature overnight. The microgel was precipitated down by centrifugation at 6000 rpm for 15 min at 4°C. The supernatant was removed carefully with a pipette and the precipitant was resuspended in 1 ml of Tris HCl, pH 8 buffer by agitating with a pipette tip and brief vortexing.
  • Example 8 heterogeneous assay with HIV-DNA and miRNA21 as target nucleotide sequence
  • Probes 1 (12 bases) specific for each target nucleotide sequence 3 and functionalised with an amine group at the 3' end were conjugated with the carboxylic groups on the surface of the microgel. Fluorophore 11 bound at the 5' of each probe 1 was Cy5. Respective probes 2 (39 bases) carrying BHQ2 as quencher 23 were hybridized to probe 1.
  • Figure 8 shows images acquired by CLSM and the intensities measured along the axis of the microgel.
  • the channel for the analysis of the code and for the detection of the displacement are shown in the lower part.
  • Figure 9A shows the recovery of fluorescence on the surface of microgels as a function of the different concentrations of the target sequence (HIV).
  • the disclosed kit allows to obtain a linear response in the emission of fluorescence in the range of concentrations between 10 -17 M and 10 -19 M.
  • the graph in figure 9b shows the linear regression of the fluorescence emissions as a function of the concentration of the target sequence in the range between 10 -17 M and 10 -19 M.
  • kit 10 for detecting a single-strand target nucleotide sequence 3 From an analysis of the features of kit 10 for detecting a single-strand target nucleotide sequence 3 according to the present invention, the advantages it allows to obtain are apparent.
  • kit 10 allows to detect target nucleotide sequences 3:
  • probes 1 and 2 by means of very specific parameters, a very high specificity can be obtained, allowing to obtain a very low aspecific signal even when complex samples with several protein species are analysed.
  • the kit according to the invention allows a very high multiplexing.
  • Kit 10 works in assays for target nucleotide sequences 3 both of DNA and RNA.
  • a high number of probes 1 can be concentrated in an extremely limited area. This allows to increase the sensitivity of the assay.
  • kit 10 The combination between multilayer microparticles 4 and kit 10 allows to:

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Claims (8)

  1. Kit (10) pour détecter une séquence nucléotidique cible à un seul brin (3), comprenant :
    - au moins une première sonde d'acide nucléique (1) de 10 à 14 bases, à l'extrémité 5' de laquelle au moins un fluorophore (11) est lié ;
    - au moins une seconde sonde d'acide nucléique (2) de 35 à 50 bases, comprenant, de l'extrémité 5' vers la 3' :
    - un premier segment (21) ayant une séquence nucléotidique complémentaire de la première sonde d'acide nucléique (1),
    - au moins un agent d'extinction (23), et
    - un second segment (22) ayant une séquence nucléotidique complémentaire d'au moins une partie de la séquence nucléotidique cible (3), dans lequel :
    - la relation suivante est satisfaite : 10 Kcal / mole > ΔG hybr . cible 3 − sonde 2 − | ΔG hybr . sonde 1 − sonde 2 > 50 kcal / mole ,
    Figure imgb0021
     où :
    - ΔG hybr. cible3-sonde2 est l'énergie libre de la formation d'un duplex entre la séquence nucléotidique cible (3) et la seconde sonde d'acide nucléique (2), et
    - ΔG hybr. sonde1-sonde2 est l'énergie libre de la formation d'un duplex entre la première sonde d'acide nucléique (1) et la seconde sonde d'acide nucléique (2) ; et
    - les concentrations de ladite au moins une première sonde d'acide nucléique (1) et de ladite au moins une seconde sonde d'acide nucléique (2) sont de 5 nM à 50 nM.
  2. Kit (10) selon la revendication 1, dans laquelle la séquence nucléotidique cible à un seul brin (3) est un ADN et 35 kcal/mole > |ΔG hybr. cible3 - sonde2| - |ΔG hybr. sonde1 - sonde2|> 45 kcal/mole.
  3. Kit (10) selon la revendication 1, dans laquelle la séquence nucléotidique cible à un seul brin (3) est un miARN et 10 kcal/mole > |ΔG hybr. cible3 - sonde2| - |ΔG hybr. sonde1 - sonde2|> 25 kcal/mole.
  4. Kit (10) selon l'une quelconque des revendications précédentes, dans laquelle ladite au moins une première sonde d'acide nucléique (1) a une longueur de 11 à 13 bases.
  5. Kit (10) selon l'une quelconque des revendications précédentes, dans laquelle la séquence nucléotidique cible à un seul brin (3) a une longueur de 15 à 100 bases.
  6. Kit (10) selon la revendication 5, dans laquelle la séquence nucléotidique cible à un seul brin (3) a une longueur de 20 à 40 bases.
  7. Kit (10) selon l'une quelconque des revendications précédentes, dans laquelle la séquence nucléotidique cible à un seul brin (3) est en une concentration de 1·10-17M à 1·10-19 M.
  8. Kit (10) selon l'une quelconque des revendications précédentes, comprenant aussi une microparticule (4) liée de façon covalente à l'extrémité 3' de ladite au moins une première sonde d'acide nucléique (1).
EP13831873.8A 2012-12-27 2013-12-27 Trousse de sonde pour détecter une séquence nucléotidique cible à un seul brin Active EP2938746B1 (fr)

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IT001154A ITTO20121154A1 (it) 2012-12-27 2012-12-27 Sistema di sonde per rivelare una sequenza nucleotidica bersaglio a singolo filamento
PCT/IB2013/061377 WO2014102748A1 (fr) 2012-12-27 2013-12-27 Trousse de sonde pour détecter une séquence nucléotidique cible à un seul brin

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NZ212419A (en) * 1984-06-25 1988-08-30 Mucan Diagnostics Pty Ltd In vitro diagnostic test for detecting cancer cells producing mucin antigens
US20030096351A1 (en) * 1998-03-27 2003-05-22 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20030108927A1 (en) * 2000-04-03 2003-06-12 Kathryn Leishman Compositions and methods for the prevention, treatment and detection of tuberculosis and other diseases
DE60213387D1 (de) * 2001-03-02 2006-09-07 Stratagene California Zusammensetzungen und verfahren mit platinverbindungen zur nukleinsäuremarkierung
US7385043B1 (en) * 2003-04-30 2008-06-10 The Public Health Research Institute Of The City Of New York, Inc. Homogeneous multiplex screening assays and kits
JP4972541B2 (ja) * 2004-04-01 2012-07-11 バイオ−ラッド ラボラトリーズ インコーポレーティッド 標識されたプローブおよび３’→５’エキソヌクレアーゼ活性を用いた定量的増幅方法
JP2008529528A (ja) * 2005-02-09 2008-08-07 ストラタジーン カリフォルニア ポリヌクレオチドの検出用のキープローブ組成物および方法
CA2723726C (fr) * 2008-05-13 2017-09-12 Michael M. Becker Oligomeres de capture de cible inactivables pour hybridation et capture selectives de sequences d'acides nucleiques cibles

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US9834812B2 (en) 2017-12-05

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